Blockade of evoked neurotransmitter release by tetanus and botulinum neurotoxins is correlated with proteolysis of their respective intracellular substrates, VAMP/synaptobrevin, SNAP-25 and syntaxin in intact neurons. Botulinum neurotoxin C cleaves syntaxin and in addition, acts directly or indirectly on SNAP-25. BoNT C is the only clostridial neurotoxin that acts on two of the three synaptic proteins that form the core complex for vesicle fusion and is cytotoxic for both young and mature neurons. Toxin blockade of synaptic vesicle exocytosis is coincident with a block in synaptic vesicle membrane retrieval, except with botulinum neurotoxin A. Synaptic vesicle endocytosis, visualized by activity-dependent uptake of FM1-43 or of horseradish peroxidase, occurs with stimulation even when vesicular release is blocked totally by botulinum neurotoxin A. The rank order of potency of the seven serotypes of botulinum in blocking neurotransmitter release in spinal cord cell cultures is D>A>B>C>G>E>F. Competitive binding studies indicate that A, B and E have different high affinity receptors. A chimeric protein consisting of the transmembrane domain of anthrax toxin and the catalytic domain of tetanus toxin inhibits (in the presence of the anthrax binding factor) endocytosis and exocytosis in nonneuronal cells, suggesting that this chimeric protein affects multiple membrane trafficking pathways.